Determination of rifabutin in human plasma by high-performance liquid chromatography with ultraviolet detection

A simple, specific and sensitive high-performance liquid chromatographic (HPLC) method was developed for the determination of rifabutin in human plasma. Rifabutin and sulindac (internal standard) are extracted from human plasma using a C₈ Bond Elut extraction column. Methanol (1 ml) is used to elute the compounds. The methanol is dried down under nitrogen and reconstituted in 250 μl of mobile phase. Separation is achieved by HPLC on a Zorbax Rx C₈ column with a mobile phase composed of 0.05 M potassium dihydrogen phosphate and 0.05 M sodium acetate at pH 4.0-acetonitrile (53:47, v/v). Detection is by ultraviolet absorbance at 275 nm. The retention times of rifabutin and internal standard were approximately 10.8 and 6.9 min, respectively. The assay is linear over the concentration range of 5–600 ng/ml. The quantitation limit was 5 ng/ml. Both intra-day and inter-day accuracy and precision data showed good reproducibility.     

Determination of benflumetol in human plasma by reversed-phase high-performance liquid chromatography with ultraviolet detection

A reversed-phase high-performance liquid chromatographic method for the determination of benflumetol in human plasma is described. Benflumetol in plasma samples was extracted with a glacial acetic acid-ethyl acetate (1:100, v/v) mixture at pH 4.0. Chromatography was performed on a Spherisorb C₁₈ column using a methanol-water-glacial acetic acid-diethyl amine (93:6:1:0.03, v/v) mixture as the mobile phase and UV-VIS detection at 335 nm. The identity and purity of the benflumetol peak were carefully examined, and the internal standard method was applied for its quantitation. The absolute recovery of benflumetol in spiked plasma samples was 92.91% over the concentration range 5–4000 ng/ml. The recovery of internal standard “8212” at a concentration of 300 ng/ml in spiked plasma was 84.85%. The detection limit of benflumetol was 11.8 ng/ml. Plasma concentration-time profiles in healthy volunteer adults were measured after a single-dose oral administration of 500 mg of benflumetol. The assay procedures were within the quality control limits.     

Determination of ticlopidine in human plasma by high-performance liquid chromatography and ultraviolet absorbance detection

A simple HPLC method has been developed for the determination of ticlopidine in human plasma. Plasma samples were buffered at pH 9 and extracted with n-heptane-isoamyl alcohol (98.5: 1.5, v/v). Imipramine was used as internal standard. Chromatography was performed isocratically with acetonitrile-methanol-0.05 M KH₂PO₄ (20:25:55, v/v) at pH 3.0 containing 3% triethylamine at a flow-rate of 1 ml/min. A reversed-phase column, Supelcosil LC-8-DB, 15 cm × 4.6 mm I.D., 5 μm particle size, was used. The effluent was monitored by UV absorbance detection at 235 nm. The method showed good accuracy, precision and linearity in the concentration range 5–1200 ng/ml. The limit of quantitation was 5 ng/ml, with a precision (C.V.) of 8.91%, which is the same as that achieved by other authors with a previously published GC-MS method. The procedure described in this paper is simple and allows the routine assessment of ticlopidine plasma concentration in pharmacokinetic studies following therapeutic doses in human subjects.     

Determination of scutellarin in rat plasma by high-performance liquid chromatography with ultraviolet detection

A validated high-performance liquid chromatography method is described for the determination of scutellarin in rat plasma using a liquid-liquid extraction and ultraviolet (UV) absorbance detection. The separation used a Diamonsil ODS column (250 mm x 4.6mm i.d., 5 microm particle size) with an isocratic mobile phase consisting of methanol-acetonitrile-50mM dihydrogen ammonium phosphate buffer (22:15:63 (v/v/v), adjusted to pH 2.5 with 1M phosphoric acid). The ultraviolet detector operated at 335 nm. Plasma samples were extracted with ethyl acetate after acidification. The extraction recovery of scutellarin ranged from 68.1 to 80.5%. High selectivity and a low quantitation limit (0.050 microg/ml) were achieved. The linear range was 0.050-12.5 microg/ml, correlation coefficient r=0.9981. The method has a good reproducibility, R.S.D. values were below 7.9% for within-day and between-day precision. The method is simple, rapid, and applicable to preliminary pharmacokinetic studies of scutellarin in rats.     

Determination of cisapride and norcisapride in human plasma using high-performance liquid chromatography with ultraviolet detection

A simple, rapid and reproducible high-performance liquid chromatographic assay for cisapride and norcisapride in human plasma is described. Samples of plasma (150 μl) were extracted using a C₁₈ solid-phase cartridge. Regenerated tubes were eluted with 1.0 ml of methanol, dried, redissolved in 150 μl of methanol and injected. Chromatography was performed at room temperature by pumping acetonitrile–methanol–0.015 M phosphate buffer pH 2.2–2.3 (680:194:126, v/v/v) at 0.8 ml/min through a C₁₈ reversed-phase column. Cisapride, norcisapride and internal standard were detected by absorbance at 276 nm and were eluted at 4.3, 5.3 and 8.1 min, respectively. Calibration plots in plasma were linear (r>0.998) from 10 to 150 ng/ml. Intraday precisions for cisapride and norcisapride were 3.3% and 5.4%, respectively. Interday precisions for cisapride and norcisapride were 9.6% and 9.0%, respectively. Drugs used which might be coadministered were tested for interference.     

Rapid quantification of indinavir in human plasma by high-performance liquid chromatography with ultraviolet detection

A rapid, sensitive and specific high-performance liquid chromatography (HPLC) procedure for the quantification of indinavir, a potent human immunodeficiency virus (HIV) protease inhibitor, in human plasma is described. Following C₁₈ solid-phase extraction, indinavir was chromatographed on a reversed-phase C₈ column using a simple binary mobile phase of phosphate buffer–acetonitrile (60:40, v/v). UV detection at 210 nm led to an adequate sensitivity without interference from endogenous matrix components. The limit of quantification was 25 ng/ml with a 0.1 ml plasma sample. The standard curve was linear across the range from 25 to 2500 ng/ml with an average recovery of 91.4%. The mean relative standard deviations for concentrations within the standard curve ranged between 1.4 and 9.7%. Quality control standards gave satisfactory intra- and inter-assay precision (R.S.D. from 3.5 to 15.8%) and accuracy within 15% of the nominal concentration. Sample handling experiments, including HIV heat inactivation, demonstrated analyte stability under expected handling processes. The assay is suitable for the analysis of samples from adult and pediatric patients infected with HIV.     

Determination of olanzapine in plasma by high-performance liquid chromatography using ultraviolet absorbance detection

A rapid method for the determination of olanzapine in plasma using high-performance liquid chromatography with ultra violet detection is described. Olanzapine was extracted from plasma with a mixture of hexane/dichloromethane (85:15), and then back extracted into phosphate buffer pH 2.8. Separation was achieved on a RP Select B C(18) column and commonly administered drugs did not interfere with the assay. The limit of quantitation was 1.5 microg/l and the inter-day and intra-day relative standard deviations were less than 10%. Olanzapine was shown to be stable in plasma for up to 7 days when stored at 4 degrees C. Moreover, the addition of ascorbic acid was not necessary for the achievement of chemical stability during storage, or during the assay procedure. The method has been used to measure olanzapine concentrations in patients treated with various doses of the drug varying from 5 to 40 mg/day.     

Determination of fluoxetine and norfluoxetine in human plasma by high-performance liquid chromatography with ultraviolet detection in psychiatric patients

A rapid high-performance liquid chromatographic method is described for the simultaneous determination of the widely used antidepressant drug, fluoxetine and its principal metabolite norfluoxetine in plasma. After liquid-liquid extraction the compounds were separated in a reversed-phase column and assayed by ultraviolet absorption at 226 nm. The analytical interference from psychoactive drugs and their metabolites was also studied. The extraction recoveries were 93 and 87% for norfluoxetine and fluoxetine, respectively. The limit of quantitation under the described conditions was 14 nmol/l for both compounds. The method was found to be reproducible with coefficients of variation less than 10%. A great variability in plasma concentrations of fluoxetine and norfluoxetine as well as in fluoxetine/norfluoxetine ratios was found among the 29 patients studied. This result suggests the implication of genetically polymorphic enzymes, presumably CYP2D6, CYP2C9 and CYP2C19 in the metabolism of fluoxetine to norfluoxetine. Therapeutic drug monitoring should thus be useful in patients treated with regular doses.     

Determination of hyperforin in human plasma using solid-phase extraction and high-performance liquid chromatography with ultraviolet detection

The role of the Mg2+ cation on antihypertensive molecule binding on human serum albumin (HSA) was studied by affinity chromatography. The thermodynamic data corresponding to this binding were determined for a wide range of Mg2+ concentrations (c). For the nifedipine molecule, an increase in the Mg2+ concentration produced a decrease in binding due to a decrease in the electrostatic interactions. For verapamil and diltiazem, which have the highest solvent accessible surface area, the solute binding on HSA was divided into two Mg2+ concentration regions. For a low c value below c(c) (approximately 1.6 mmol/l), the binding dependence with c was similar to that of nifedipine. For c above c(c) the hydrophobic effect created in the bulk solvent associated with a decrease in the van der Waals interactions between the solute molecule and the HSA implied a decrease in its binding. These results showed that for patients with hypertension, an Mg2+ supplementation during treatment with these antihypertensive molecules can increase the active pharmacological molecule concentration.     

Determination of celecoxib in human plasma by normal-phase high-performance liquid chromatography with column switching and ultraviolet absorbance detection

A method is described for the determination of celecoxib in human plasma. Samples were extracted using 3M Empore membrane extraction cartridges and separated under normal-phase HPLC conditions using a Nucleosil-NO₂ (150×4.6 mm, 5 μm) column. Detection was accomplished using UV absorbance at 260 nm. The HPLC method included a column switching procedure, in which late eluting compounds were diverted to waste, to reduce run-time to 12 min. The assay was linear in the concentration range of 25–2000 ng/ml when 1-ml aliquots of plasma were extracted. Recoveries of celecoxib were greater than 91% over the calibration curve range. Intraday precision and accuracy for this assay were 5.7% C.V. or better and within 2.3% of nominal, respectively. The assay was used to analyze samples collected during human clinical studies.     

Determination of dihydroergotamine in human plasma by high-performance liquid chromatography with fluorescence detection

Dihydroergotamine, a 5-hydroxytryptamine antagonist, is used for the treatment of vascular headaches. A high-performance liquid chromatography assay with fluorescence detection is described for the determination of dihydroergotamine in plasma. The assay was validated over the concentration range 0.1–10 ng/ml plasma and applied to the analysis of plasma samples from subjects treated intramuscularly and intranasally with 2 mg of dihydroergotamine.     

Determination of succinylcholine in human plasma by high-performance liquid chromatography with electrochemical detection

An alternative HPLC method for the quantification of the depolarizing neuromuscular blocking agent succinylcholine in human plasma is described. Drug spiked plasma and patient plasma samples were extracted using a C₁ solid-phase cartridge. Succinylcholine was separated on a Cyano column and quantitated using electrochemical detection at a potential of 450 mV and 750 mV. Mobile phase consisted of a mixture of phosphoric acid–acetonitrile–methanol (45:35:25) adjusted to an apparent pH of 5. Standard curves for the quantitation were linear in the range of 250–8000 ng/ml. Between-day and within-day relative standard deviations were 5.1% and 1.7%, respectively. Mean drug recovery and accuracy was 68% and 104%, respectively.     

Determination of amiprilose in human plasma by high-performance liquid chromatography with fluorimetric detection

A reversed-phase HPLC method to quantify amiprilose in human plasma is described. The method involves liquid–liquid extraction of amiprilose and the internal standard from plasma. The extracted compounds are derivatized with 1,8-naphthalic dicarboxylic acid using 2-chloro-1-methylpyridinium iodide as a coupling reagent. The derivatized products are separated on a reversed-phase column and monitored fluorimetrically using 280 nm and 340 nm as excitation and emission wavelengths, respectively. The derivatized products which exhibit two peaks on chromatogram, are shown to be the interconvertible isomers. This assay has been used in pharmacokinetic studies of amiprilose in humans.     

Determination of naloxone in human plasma by high-performance liquid chromatography with coulometric detection

Naloxone, the analyte and the internal standard, sumatriptan, are extracted from plasma using solid-phase extraction columns. Chromatography and detection are performed using isocratic reversed-phase high-performance liquid chromatography (HPLC) with coulometric end-point detection. The standard curve was linear over the range 0–50 ng/ml of naloxone in plasma. The reproducibility, the coefficient of variation (C.V.) of the method over the range of the standard curve was 6.2–11.2%. The recovery averaged 90.4±8.9%. A plasma profile following i.v. administration of naloxone in one normal healthy volunteer is presented.     

Determination of metformin in human plasma by high-performance liquid chromatography with spectrophotometric detection

A simple, selective, sensitive and precise high-performance liquid chromatographic plasma assay for the hypoglycemic agent metformin is described. Acidified samples of plasma were deproteinated with acetonitrile, washed with dichloromethane and the resulting supernatant injected. Chromatography was performed at 40°C by pumping a mobile phase of acetonitrile (250 ml) in pH 7, 0.03 M diammonium hydrogen phosphate buffer (750 ml) at a flow-rate of 1 ml/min through a silica column. Metformin and the internal standard (atenolol) were detected at 240 nm and were eluted 7.8 and 6.8 min, respectively, after injection. No endogenous substances were found to interfere. Calibration curves were linear (r>0.999) from 10 to 2000 ng/ml. The absolute recovery of both metformin and atenolol was greater than 76%. The detection limit and limit of quantitation were 2.5 and 10 ng/ml, respectively. The intra- and inter-day precision (C.V.) was 12%, or less, and the accuracy was within 6.2% of the nominal concentration. This method is suitable for clinical investigation and monitoring metformin concentration.     

Determination of amlodipine in human plasma by high-performance liquid chromatography with fluorescence detection

A sensitive and specific HPLC method has been developed for the assay of amlodipine in human plasma. The assay involves derivatization with 4-chloro-7-nitrobenzofurazan (NBD-Cl), solid-phase extraction on a silica column and isocratic reversed-phase chromatography with fluorescence detection. Nortriptyline hydrochloride was used as an internal standard. The assay was linear over the concentration range of 0.25–18.00 ng/ml. Both of the within-day and day-to-day reproducibility and accuracy were less than 11.80% and 12.00%, respectively. The plasma profile following a single administration of 10 mg amlodipine to a healthy volunteer was presented.     

Determination of catecholamines in human plasma by high-performance liquid chromatography with electrochemical detection

In the present study, assays were improved for the determination of catecholamines in human plasma. High-performance liquid chromatography with electrochemical detection was employed for quantitative analysis. The influence of various parameters on chromatographic performance, such as the composition and the pH of the mobile phase, and the detection potential, was investigated. An accurate solid-phase extraction procedure, after catecholamine complexation with diphenylborate, was developed. The efficiency yield for all catecholamines was in the range 92–98%. Relative standard deviation values for repeatability and for intermediate precision were less than 2% and 3%, respectively, for all three analytes.     

Determination of loratadine in human plasma by high-performance liquid chromatographic method with ultraviolet detection

A HPLC–UV determination of loratadine in human plasma is presented. After simple liquid–liquid extraction with 2-methylbutane–hexane (2:1) and evaporation of organic phase the compounds were re-dissolved in 0.01 M HCl, evaporated again and finally separated on a Supelcosil LC-18-DB column. The analyses were done at ambient temperature under isocratic conditions using the mobile phase: CH₃CN–water–0.5 M KH₂PO₄–H₃PO₄ (440:480:80:1, v/v). UV detection was performed at 200 nm with a limit of quantification of 0.5 ng/ml. The precision was found to be satisfactory over the whole range tested (0.5–50 ng/ml) with relative standard deviations of 2.3–6.3 and 5.2–14.1% for intra- and inter-assays, respectively.